Washing process and centrifuge assembly

ABSTRACT

A fluid system for separating materials in each of a plurality of batches which are simultaneously washed by forcing a fluid through a centrifuge, provides independent pressure heads for each batch to enable flow of equal volumes through said batches, respectively.

I United States Patent m1 3,561,672

[72] Inventors Charles A. Schlutz [56] References Cit d g k Ch I UNITEDSTATES PATENTS z 'g g fgf ffi 2,906,453 9/1959 Driscoll 233/28 A I No595 3,244,363 4/l966 Hein 233/26 [22] fg 18 1968 3,347,454 10/1967Bellamy... 233 17 9 [45] Patented Feb.9, 1971 Primary ExaminerRobert W.Jenkins [73] Assignee Baxter Laboratories, Inc. Attorneys-Walter C. Kehmand Richard J. Reilly Morton Grove, III. a corporation of DelawareWASHING PROCESS AND CENTRIFUGE ASSEMBLY 22 Claims, 12 Drawing Figs.

US. Cl 233/17, 233/ l 9 Int. Cl B04b 5/02 Field of Search 233/17, 26,

ABSTRACT: A fluid system for separating materials in each of a pluralityof batches which are simultaneously washed by forcing a fluid through acentrifuge, provides independent pressure heads for each batch to enableflow of equal volumes through said batches, respectively.

PATENIEIDVFEB 9mm SHEET 1 OF 4 INVE snows CHARLES A. ScHLur STANLEY J.SEDIVY CHARLES R. Msmmnor J, A w.

PATENTEU FEB 9 l97| SHEET 3 OF 4 I62 INVENTOR$ CHARLES A.SCHLUTZ.

STANLEY J. SEDW CHARLES R. MEMHAROT 521, W}.

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SHEET UF 4 EEREG 5:550 E INVENTORS CHARLES A. ScHLuTz STANLEY J. SEDWYCHARLES R.MEMHAROT 1 Wy. W A'r'rv.

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PATENTEU FEB 9 I97! l WASHING PROCESS AND CENTItIFUGE ASSEMBLY Thepresent invention relates to centrifugation. More particularly theinvention relates to centrifugal apparatus for use in washing.Specifically the invention relates to an assembly, including acentrifuge, for washing particles such as red b!ood cells.

Heretofore it has been known to wash particles by using centrifugingmeans. The principles generally relating to such particle washing havebeen effectively applied in washing red blood cells, particularly forreconstitution following treatment with such reagents as glycerol,polyvinyl pyrrolidone and the like for cell protection. Two heretoforeknown systems for reconstituting preserved red blood cells by theaforestated technique have gained significant practical success. One

thereof embodies the teachings of US. Pat. 2,906,453. The

other thereof embodies the teachings of US. Pat. 3,347,454 and isassigned to the assignor of the present application.

While the apparatus of both prior systems are practical for washingpreserved red blood cells in' single batches, neither they, nor any ofheretofore known means, is adequate effectively and efficiently forwashing av plurality of batches of preserved red blood cellssimultaneously. That is to say, while simultaneous multibatch washingcould be practiced in ac cordance with heretofore known teachings,thereto attendant inefficiencies would increase costs inordinately. Inconsequence, prior to the present invention, washing of red cells inmore than one batch at a time in a given centrifuge assembly has beenneither practical no'r' conventionally practiced, except experimentally.The inefficiencies result from differential resistance to flow of washfluid generated in material comprising the batches 7 simultaneouslybeing processed. The phenomenon is such that wash fluid in everincreasing volume flows throughone of the batches while it increasinglybypasses any other.

It is an object of the present invention to provide an improved processand assembly for centrifugal washing.

It is another object of the invention to provide improved process andapparatus for centrifugally washing particles, particularly, red bloodcells.

It is a further object of the invention quickly and efficiently toreconstitute red blood cells which have been treated with cryoprotectiveagents.

It is an additional object of the invention to enable washing of aplurality of batches of particles, particularly red blood cells,simultaneously. J

Moreover, it is an object of the invention to prevent inefficientmaterial separation resulting from differential resistance to flow in aplurality of batchesduring simultaneous washing thereof in a centrifuge.

The foregoing objects are effected, in a washing centrifuge assemblyhaving a rotor and a pair of containers supported from said rotor forrotation therewith, by charging means for moving influent into each ofsaid containers under an independent pressure head as said rotor spins,and discharging means for collecting effluent from each of saidcontainers in a volume corresponding to influent moved into saidcontainers. In another sense the objects are effected by a process inwhich material to be washed is separated into a plurality of batcheswhich are simultaneously centrifugally spun. While they are spinninginfluent under the force of an independent pressure head is flowedthrough each of the batches; and effluent from said batches is collectedin volumes corresponding to the volumes of influent flowed through saidbatches.

The foregoing and other objects, features and advantages of theinvention will become more apparent upon consideration of the followingdescription and appended claims, when considered in conjunction with theaccompanying drawings wherein the same reference character or numeralrefers to like or corresponding parts throughout the several views.

On the drawings:

FIG. I is a fragment of a vertical sectional view through a centrifugeassembly embodying the present invention, some parts being shown inelevation and other parts broken away, a

pair of containers being shown at an angle 90 from actual aspect and ina nonspinning condition.

FIG. 2 is a flow scheme of said assembly, the containers being shown ina spinning condition.

FIG. 3 is a top plan view of a distributor-collector comprising saidassembly.

FIG. 4 is a vertical sectional view according tothc line 4-4 of FIG. 3.

FIG. 5 is a vertical sectional'view according to the line 5-5 of FIG. 3.

FIG. 6 is a horizontal sectional"viewaccording to the line 6-6 of FIG.4.

FIG. 7 is a top plan view of an assembly control box, its cover havingbeen omitted.

FIG. 8 is a view according to the line 8-8 of FIG. 7 but with the covershown.

FIG. 9 is a side elevation view of fluid control means comprising saidassembly, a pair of clamping members being shown abnormally in oppositeconditions.

FIG. 10 is a view according to the broken section line I0- I0 of FIG. 9.

FIG. 11 is a view according to the line 11-" of FIG. I, a medial partbeing broken away.

FIG. 12 is a scheme of wiring for said assembly.

Referring now more particularly to FIG. I, there is shown a washingcentrifuge assembly I5 comprised of a housing or outer shell I7 only anupper portion of which is shown. Within chamber 19 of shell 17 acentrifuge rotor 21 is concentrically disposed for rotation. Rotor 2Ihas a hub 25 which is mounted corotationally with a driven spindle 27,the latter member being suitably connected to prime mover means (notshown). A plurality of radial members 26 (only two of which are shown)project outwardly from hub 25. A plurality of arms 29,

arranged in parallel pairs, project ecccntrically from adjoining 1 ofmembers 26. A pin 33 which is circular in cross section extends througheach of arms 29 for projection from opposite arm faces 31. Adjoiningprojections of pairs of pins 33 are engaged by opposed pairsof arcuateshoulders 35 of a plurality (herein shown as a pair) of receptacles 23and 24 for swinging support of said receptacles radially from rotor 21.Apart from evident structural variation, said receptacles may beconstructed according to copending application Ser. No. 672, l 25,assigned to the assignee of the presentapplication.

Container means comprising aplurality of containers 37 and 38, which maybe fashioned according to said copending application, are disposed inreceptacles 23 and 24, respectively, in a manner such that eachcontainer is supported from said rotor for rotation therewith. Materialto be processed, herein shown as particles 36 such as preserved redblood cells, is divided into a plurality of batches which may be ofequal size, and held in containers 37 and 38 for spinning forcentrifugation simultaneously with rotation of rotor 21. The invention,however, is not limited to washing simultaneously only one pair ofbatches. More can be washed simultaneously. Nor is the invention limitedto washing particles, inasmuch as washing of materials in other physicalforms is also comprehended.

In the illustrated embodiment, an inverted cone-shaped base provides aseat 39 in each of the receptacles 23 and 24 I for an associated ofcontainers 37 and 38. A cap 4! for each of receptacles 23 and 24 hasapair of bores through which an influent duct 43 and an effluent duct 45extend for fluid circulation through each of said containers. The partsare proportioned so that a flange 47 on each of said caps will snuglyseat against a rim 49 of a corresponding receptacle as a result ofcentrifugal force when the parts assume the relationship shown in FIG.2. However, when receptacles 23 and 24 are in the condition of FIG. Ithe normal pressure under which each container is filled with itsmaterial to be washed may hold each flange 47 spaced from associated rim49.

Fluid control mean responsive to centrifugal force comprises acentrifugal clamp assembly generally designated SI (FIGS. I, 9 and 10)which is secured by fasteners such as screws 53 to the top of hub 25 forcorotation with rotor 21.

Said fluid control means has a first condition in which ducts 43 and 45are shut to block flow into and out of containers 37 and 38 and a secondcondition in which said ducts are open for flow into and out of saidcontainers. To that end, clamp assembly 51 comprises a body 55 having aplurality of wells 56 and 57 which are herein shown of like profile anddisposed to correspond with the disposition of containers 37 and 38,herein shown as being in diametric opposition. Clamping means comprisinga clamping mechanism 58 associated with each of containers 37 and 38 isdisposed in each of wells 56 and 57, respectively. A block 59 compriseseach clamp mechanism 58 and is proportioned to slide centrally andoutwardly within the limits of its associated well along a floor 60defining such well. A cover 93 (FIG. 9) suitably removably held on body55 closes wells 56 and 57 from above to trap in each thereof anassociated block 59.

Each block 59 has a plurality (herein shown as two) of outwardlyopening, horizontally spaced apart bores 61 and 62 which accommodatespring means comprising a pair of compression springs 63 and 64,respectively. The outer ends of each pair of the latter members bearagainst the inner surface 65 of an associated of outer sidewalls 68 and70 respectively defining wells 56 and 57 for biasing each block inwardlyor centrally. Each block 59 carries a generally T-shaped clamp member 67which is secured thereto by a suitable fastener such as a screw 69. Thestem 71 of each clamp member comprises a radial arm which is slideablyarranged for radial extension in a slot 73 in body 55, said slotsfashioned so that wells 56 and 57 open through sidewalls 68 and 70. Eachclamp member 67 also comprises a pair of clamping extensions 75 and 76integral with, and projecting horizontally in opposite directions from,an associated stem 7. Under normal tensioning of each pair of springs 63and 64 its clamping extensions are urged toward an adjoining ofsidewalls 68 and 70,.as illustrated to the right of FIGS. 9 and 10. Saidsprings, however, are tensioned so that under centrifugal forcegenerated when receptacles 23 and 24 have assumed the extreme positionof FIG. 2, its block 59 will be thrown outwardly to the outer limits ofits recess 57 thereby causing projection of corresponding clampingextensions 75 and 76 from the body 55, as illustrated to the left ofFIGS. 9 and 10. It is appreciated that all of the clamping mechanisms 58simultaneously will assume the condition shown either to the right or tothe left of FIGS. 9 and and that the showing of both conditions in eachview is solely for the purpose of illustrating alternate extremes, whichnormally will notbe present simultaneously.

Each pair of extensions 75 and 76 is fashioned to generate a pair ofrecesses or pockets 77 and 78 through which influent and effluent ducts43 and 45 of an associated of containers 37 and 38 project. Each pair ofsaid recesses is proportioned so that when an associated clamp member 67is in its first condition, as shown to the right of FIGS. 9 and I0, flowin corresponding ducts 43 and 45 will be clamped shut. Whereas, whensuch clamp member is in its second condition, illustrated to the left ofFIGS. 9 and 10, said last mentioned ducts will be unclamped to permitunimpaired flow therethrough.

To facilitate clamping, each of sides 68 and 70 has a pair of horizontalelevations 81 and 83 .which project in opposite directions from anassociated slot 73. At its outer end each of extensions 75 and 76 has apair of enlarged ears 85 and 87 which are associated in a U-shape andproportioned for engagement about associated of elevations 81 and 83 ina manner illustrated to the right of FIG. 9 for clamping correspondingducts 43 and 45. l

Distributor means or a fluid handling mechanism comprising adistributor-collector 89 controls flow of fresh influent or wash fluidto and spent effluent or waste from the material contained in containers37 and 38. In terms of the influent, distributor-collector 89 isdisposed upstream of clamp assembly 51 from which it projects upwardly.The distributor collector comprises relatively rotatable first andsecond parts, being a cup-shaped rotor 97 and a therein concentricallyassociated cylindrical stator 105 (FIG. 4).

A male component (FIGS. '1, 4 and 5) carried from the lower wall 99 ofrotor 97 is removably keyed or snugly en' gaged in an opening or hole 91(FIG. 10) formed in body 55 for transmission of torque of rotor 21 to'rotor 97 for corotation of said rotors. Rotor 97 has an upwardlyextending cylindrical sidewall 107 which defines an upper opening 108.The latter is closed by a cap IOI-having an aperture 109 and secured tothe top of sidewall 107 by fasteners such as screws 103, only one ofwhich is numbered in FIG. 3.

The outside diameter of stator is less than the inside diameter of rotor97 by reason of which a space I13 is formed about said stator, saidspace being closed at the top and bottom by cap 101 and wall 99,respectively. Said stator has a lower end portion 106 which is journaledin wall 99, and a medial portion which is journaled in the aperture ofcap 101 and an enlarged head 111 which projects upwardly from the rotorand is arranged in bearing association with the top of cup 101.

Chamber defining means comprise a plurality of annular flexible sealingmembers 115, 117, I19 and 121 which are arranged in vertically spacedhorizontal planes with the upper sealing member 121 disposed against cap101 for dividing space 113 into a plurality of vertically spacedchambers I23, 125, 127 and 129. Thecircumferences of the sealing memberssnugly engage the inner surface of rotor wall I07 while the innersurfaces of said sealing member snugly engage the outer surface ofstator 105. Thereby each of chambers I23, I25, 127 and 129 is sealedfrom the others thereof and is defined by an inner wall comprising asection of stator I05 and an outer wall comprising a section of rotor97. A pair of apertures in the chamber walls comprise inlet and outletmeans for said chambers and accordingly distributor-collector 89.

In the present embodiment each of the receptacles 23 and 24 isassociated with a pair of said last mentioned chambers in a manner suchthat one of each pair is an influent chamber and the other thereof is aneffluent chamber. In the present and preferred embodiment of theinvention the lowermost chambers I23 and are influent chambers of whichchamber 123 is associated with receptacle 24, whereas chamber 125 isassociated with receptacle 23. To that end, the upstream end portion 129of duct 43 from receptacle 24 projects through one outlet opening inwall 107 in a fluid tight seal for fluid communication with chamber 123.On the other hand, the upstream end portion 131 of duct 43 fromcontainer 23 extends through the other outlet opening in wall 107 in afluid tight seal for fluid communication with chamber 125. In thepresent and preferred embodiment, chambers I27 and 129 are effluentchambers which are disposed above influent chambers 123 and 125 and arein fluid communication, respectively, with receptacles 23 and 24. Forthat purpose the upper end portion 133 of duct 45 associated withreceptacle 24 projects through one inlet opening in wall 107 in a fluidtight seal for fluid communication with chamber 127. The upper endportion 1350f duct 45 associated with receptacle 23 projects through theother inlet opening in wall 107 in a fluid tight seal for fluidcommunication with chamber 129.

By placing influent chambers 123 and 125 below the effluent chambers,the likelihood of contamination of influent by effluent because offaulty seals in distributor-collector 89 is minimized. Moreover, thepressures generated within the system are such that downstream ofcontainers 37 and 38 the effluent paths may be combined, if desired,with impunity.

Stator 105 has a plurality of vertical bores 137, 139, I41

and 143 (FIGS. 4, 5 and 6) which open through the top of stator head11]. The lower ends of bores 137, 139, 141 and I43, respectively,communicate with chambers 123, I25, 127 and 129. By reason of theforegoing, bores 137 and 139 are influent bores whereas bores 14] and143 are etfluent bores.

Charging means for moving influent or wash fluid into containers 37 and38 and through the batches therein contained comprises a pump 153 (FIG.2) which is arranged in fluid communication with influent bores 137 and139 through a pair of flexible conduits 145 and147. Said conduitstogether with distributor-collector 89 and ducts 43, 43 comprise meansdefining an independent path or influent pathway for each of containers37 and 38 and extending between said containers and the pump. Pump 153may be of the roller type for impelling substantially equal volumes ofinfluent under independent pressure heads into conduits 145 and 147 andaccordingly containers 37 and 38. In the present embodiment, movement ofinfluent through each system is simultaneous; however other thansimultaneous influent movement may be desirable under washing conditionsother than that specifically described herein.

Fluid from a reservoir 233 supplies 'influent for pump 153 by gravity.The reservoir is also in direct fluid communication with conduits 145and 147 through a pair of tubes 235 and 237, which bypass the pump forgravity feed into containers 37 and 38, respectively, for a reason tobecome apparent from ensuing description.

Discharging means for collecting effluent or spent wash fluid comprisesan effluent tank 155, which is in fluid communication with bores 141 and143 through duct means herein comprised of ducts 149 and 151,respectively.

A bearing mechanism generally designated 157 (FIGS. 1 and 11) for therelatively stationary part of a rotational assembly comprises a retainer159 having a pair of spaced apart fingers 161 and 162 which define anelongated recess 163 in which an upper reduced portion 165 of statorhead 11] is intimately engaged for sliding normally of an expansion bandor bar 167 when the latter is in assembled condition. The retainer iscaptured in a pocket 166 formed in medial section 168 of expansion bar167 to permit sliding of the retainer longitudinally of said bar inopposite directions in response to corresponding stresses duringrotation of rotor 97.

Bar 167 comprises a pair of opposed end sections 158 and 160 at leastone of which is telescopically connected to medial section 168. Said endsections carry a pair of opposed fittings or adapters 169 and 177 havingoutwardly extending hooks 171 and 181, respectively for projection intoa mating aperture formed in the lower end of a skirt 173 (FIG. I) hungin the top opening of the centrifuge from shell 17. An annular cushionor pad 175 is shown disposed between the said shell and skirt 173 forcushioning evident forces brought to bear. In consequence of theforegoing construction, distributor-collector 89 will be held erectduring rotation of rotor 21, fingers 161 and 162 being proportioned toprevent removal thereof. However, bar 167 may be contracted to disengagehooks 171 and 181 from skirt 173 for removing said distributor-collectorfrom the centrifuge. Thereafter, release of the rotor 97 from bar 167 byremoving the stator head from recess 163 can be achieved.

1n the illustrated embodiment section 158 comprises a vertical plate 185with which hook 171 is integral and a vertical plate 189. The inner endportions of said plates are arranged in sliding association with theoutside of opposed parallel faces of medial section 168. The outer endportions of said plates are separated by a spacer 187 the width of whichis equal to the width of section 168 from the proximate end of whichsaid spacer is spaced. To one face of said spacer, plate 185 is securedby fasteners such as rivets 179 and to the opposite face of said spacer,plate 189 is secured by fasteners such as rivets 191. One end portion ofmedial section 168 has a well 193 in which one end of a compressionspring 195 is mounted for extending block 187 and accordingly fitting169 longitudinally of bar 167 and for holding said bar in mountedposition in the centrifuge.

in addition to the condition shown in H6. 11, bar 167 can assume twoother positions according to the condition of latch means 197 which ismounted transversely of said bar from an end portion 199 of section 168.Said latch means comprises a part in he form of a rod 201 which extendsthrough a transverse 0;- ming or slot 223 in section 168 and a pair oftransversely aligned apertures 203 and 205 in telescopic section 158.While the dimension of opening 223 longitudinally of section 168 isconsiderably larger than the diameter of rod 201, the

diameter of apertures 203 and 205 is such that latch means 197 is snuglyengaged to limit its movement in section 158 to transverse sliding. Thediameter of aperture 205, however, is larger than aperture 203 toaccommodate sliding of another part of said latch means, said other partbeing an annular bearing extension 206 of a bearing cap 207. The latteris secured by a screw 209 on one end portion 211 of rod 201 whichprojects normally from the outer face of plate 189. A spring retainer213 in the form of a cup is secured by a screw 215 to the opposite endportion 217 of rod 201 said opposite end portion projecting normallyfrom plate 185. A compression spring 219, which is disposed about endportion 217, has one end disposed in retainer 213 and the other endbearing against vertical plate 185 for drawing cap 207 into engagementwith the outer surface of plate 189. The parts are proportioned so thatunder normal biasing bearing extension 206 will be drawn inwardly ofplate 189 into a reduced part of opening 223.

That is to say, opening 223 has a pair of parallel limiting walls 225and 227 spaced apart longitudinally of section 168. These walls definethe limits of movement of rod 201 longitudinally of bar 167. Within therecess 223 there is generated adjacent plate 189 annular shoulder 229within the annulus of which bearing extension 206 is normally biased.The diameter of the annulus formed by the shoulder 229 is less than thespace between walls 225 and 227 so that while the parts are conditionedas in FIG. 11 contraction of bar 167 will be limited by engagement ofthe extension 206 and the shoulder 229. The parts are proportioned sothat this extent of contraction is not enough to permit removal of bar167 from the centrifuge. However; it is adequate for accommodatingvariation in the distance across (herein shown as a diametric cord)skirt 173 and provided for bar 167 in assembled condition.

Yet another condition of the bar 167 is required for removing it fromthe centrifuge. This is achieved by pushing rod 201 upwardly withrespect to FIG. 11 against the bias of spring 219 until cup 213 engagesplate 205. The parts are proportioned so that upon the last occurrence,extension 206 will be removed from recess 223. Accordingly, rod 201 willbe free for engaging wall 225. in such last condition bar 167 will havebeen contracted sufficiently to permit its removal from the centrifuge.

The foregoing combination enables economical employment of a disposablecollector-distributor 89 for each washing operation whereby likelihoodof introduction of contaminants may be minimized.

Flow through the system defined by passaged means comprising ducts 43,45, 149 and 151 and conduits and 147 is controlled through electricalcircuitry (FIG. 12) comprising three solenoids 239, 241 and 243. Thelatter together with said circuit are arranged within a control box 245(FIGS. 7 and 8) having an upper horizontal plate 247 relative to which acover plate 249 is hingedly arranged for seeurance by a latchingassembly 255. in the present embodiment, the latching assembly comprisesa post 257 which projects upwardly from plate 247 through a registeringaperture in the cover plate 249 so that adjoining flat surfaces of saidplates may be releasably held in engaged association by a lock 259associated with said post.

Plate 247 has a plurality of upwardly opening channels or grooves 261,263 and 265. Pressure feet or pads 267, 269 and 271 carried on the endsof reciprocating arms controlled by solenoids 239, 241 and 243respectively, are arranged for extension through apertures providedtherefor in the bottoms of said channels, respectively. Medial portionsof tubes 235 and 237 are disposed in channel 261. Downstream portions ofducts 149 and 151 are disposed in channel 263. Portions of conduits 145and 147, which are upstream of their communication with tubes 235 and237, are disposed in channel 265. The parts are arranged andproportioned so that when the solenoid is deenergized the flow pathsthrough their associated tubes, ducts and conduits are open. However,while plate 249 is closed (its normal operating condition) and asolenoid is energized, its pressure foot will be projected to clamptherewith associated ducts, conduits and tubes to cut their flow paths.

For operating the device a plug 273 (HQ 12) for connecting the powercircuit 275 of the assembly to a l l volt AC power source (not shown) isprovided. The primary of a transformer 277 is arranged in series inpower circuit 275 which becomes energized upon closure of apush-to-make, push-tobreak, double pole switch 279. in the presentembodiment of the invention, the secondary of transformer 277 is a 24volt coil with which a selenium rectifier 281 is arranged in parallelfor developing a DC effect. Simultaneously with the energization oftransformer 277, a lamp 3135, which may be carried from a switch box307, will light by reason of connection to one terminal 237 of rectifier231 through a conductor 309 and thereby indicate the condition of thepower circuit 275. The other side of said rectifier is connected to aplane of reference potential, herein shown as ground 282.

One terminal of each of solenoids 239, 241 and 243 is connected througha conductor 283 to ground. The other terminal 285 of solenoid 239 isconnected to terminal 287 of said rectifier through a switch 289 of anormally closed relay 291. The other terminal 293 of solenoid 241 isconnected to terminal 287 of said rectifier through a switch 295 of anormally cl Jsed relay 297. The other terminal 299 of solenoid 243 iscmnected to terminal 287 of said rectifier through a switch 301 of anormally closed relay 303. By reason of the foregoing construction, oncethe plug 273 has been connected to a power source and the switch 279closed, solenoids 239, 241 and 243 will become energized whereuponpressure feet or pads 267, 269 and 271 will be conditioned for cuttingflow in the system.

A grounded coil 309 (upper left of FIG. 12) of a normally closed relay311 is energized upon closure of switch 279 by reason of a conductor 313which connects one side of said last mentioned coil and terminal 285whereupon switch 315 of relay 311 will open. Relay 311 is arranged forlighting a lamp 317 by reason of a circuit wire 319 which will connectsaid last lamp to the selenium rectifier through a conductor 318 whenswitch 315 is in closed condition.

To commence operation of the centrifuge, a normally open switch 321which is mounted in a housing 324 is manually depressed momentarily toclosed condition. Switch 321 is connected in parallel with the startingswitch of the circuit of a centrifuge motor or prime mover means fordriving spindle 27 in a manner such that once closed, said circuit willcontinue to operate the centrifuge regardless of the condition of switch321 until stopped by means to be hereinafter described. Said motor maybe of conventional construction and accordingly it together with itscircuit have been omitted in the drawings.

Relay 291 is included in multiple assemblies 323 which also comprises anormally open relay 325 and a common coil 327. The latter member isconnected to the circuit (not shown) of the centrifuge motor. Thereby,upon closure of switch 3211, switch 329 of relay 325 and switch 239,which are both magnetically coupled to the coil 327, respectively, willclose and open. When the switch 239 opens, current flow to solenoid 239as well as to coil 3119 become interrupted. Thereupon pressure foot 267will retract to open tubes 235 and 237 for establishing fluidcommunication between reservoir 233 and containers 37 and 38, providedrotor 21 is spinning fast enough for clamp assembly 51 to have opened.in consequence thereof the system will fill with lnfluent and initialflow through the system will be of gravity. Also, switch 315 will assumeits normally closed condition causing lamp 317 to light, therebyindicating the open condition of tubes 235 and 237 in control box 245.Simultaneously, because one terminal 331 of relay 32.5 is connected toterminal 237 while the other terminal 333 of said last relay isconnected to a grounded lamp 335 mounted in housing 324 through acircuit conductor 337, lamp 335 will light as an indicator of theoperating condition of the centrifuge.

The precaution of preventing initial filling of the system until apredetermined centrifugal force is reached tends to prevent backflowfrom containers 37 and 38 and possible contamination of influent.Moreover, accidental emptying of con' tainer contents is also preventedby thus keeping the system closed.

Stopping of gravity flow of influent is effected by normally openpush-to-make, push-to-break, double pole switch 339 which is mounted ina housing 341. Switch 339 may be operated manually as shown herein or inresponse to an electrical effect generated in a known manner in responseto a condition such as a lapse of time or color or density of effluentaccumulating in ducts 149 and 151. Once closed, switch 339 will be heldin closed condition by reason of magnetic coupling to a coil 345 whichis mounted in housing 341. Said coil is immediately energized on closingof switch 279 through a relay 347 whose switch 349 is normally biased toclosed condition and which is connected to terminal 287 through acircuit wire 351. A voltage reducing resistance 343 is interposed in aconductor 353 which connects coil 345 to relay 347.

A circuit wire 355 connects one terminal 357 of switch 339 to a groundedlamp 360, which when lit indicates operation of pump 153 and pumpgenerated flow through the system. Said terminal 357 is arranged forconnection, through said last switch when closed to rectifier terminal287 through a circuit conductor 359. The other terminal 361 of switch339 is connected to one side of a grounded relay coil 363. Coil 363 isdisposed in a housing 367 for magnetic coupling to an assembly of relayscomprising heretofore described relays 297 and 303 and additional relays369 and 371. Switches 373 and 375 of relays 369 and 371 are normallybiased to an open condition. The arrangement is such thatwhen coil 363is energized switches 373 and 375 will be magnetically moved to closedcondition whereas switches 295 and 301 will be opened.

Upon opening of switches 295 and 301 circuits for solenoids 241 and 243are opened and said last solenoids accordingly become deenergized.Thereupon pressure feet 269 and 271 move to nonclamping condition andfluid communication becomes established between pump 231 and containers37 and 38 and between said containers and effluent tank 155.Simultaneously, solenoid 239 becomes energized by reason of closure ofrelay 369. Accordingly pressure foot 267 moves to closed condition tointerrupt flow in tubes 235 and 237. The last result is effected becauseone terminal 377 of relay 369 is connected to terminal 287 of rectifier281 through a circuit wire 379 and the other terminal 381) of said relay369 is connected to the plane of reference potential through a circuitsembly including conductor 381. The latter connects relay 369 to aterminal 383 of a relay 385. in turn terminal 383 is connected by acircuit wire 337 to a terminal 389 of relay 289. However, relay 289 isbypassed by a circuit wire 391 which connects terminal 389 to terminal285 of solenoid 239 for connection of the latter through bypass 391 torelay 369. in consequence of the foregoing, when relay 369 is closed thecoil 3119 of relay 315 will also become energized to magnetically impelswitch 315 to an open condition thereby to extinguish lamp 317simultaneously with the closure of tubes 235 and 237. When solenoids 2Mand 2413 are energized, pump 153 is also actuated by reason of closureof relay 371 which is serially connected between a pair of l 15 volt ACcircuit wires 393 and 395 comprising the circuit of said pump.

For operation, the pressure of pump 153 is set at a level sufficient toovercome the centrifugal force created and to effect a flow throughcontainers 37 and 38 for washing material 36 and delivering effluent totank 155 corresponding to the influent volume. The rate of flow can becontrolled in accordance with requirements and factors including thecharacter of the material being washed. None of these factors iscritical to the present invention and accordingly details of the processdepending upon the character or type of material are omitted.

Suffice it to say that washing may be stopped at any desired time byclosing a double pole, push-to-malte, push-to-brealt switch 397 which ismounted in a housing 399. Said last switch may be manually operated (asillustrated) or automatically in response to an effect generated bywell-known means according to lapse of time or a physical characteristicof the effluent. Once closed, switch 397 may be held in closed conditionby the magnetic force induced about a grounded coil 401 which from aterminal 403 is connected to heretofore defined circuit wire 359 througha voltage lowering resistance 405. However, switch 397 can also bereleased by breaking the circuit to coil 40] by depressing a normallyclosed interruptive switch 407 disposed in the circuit of said lastcoil.

Upon initial closure of switch 397 both of its terminals 409 and 411become operatively connected to rectifier terminal 287 through circuitwire 359. in turn terminal 409 is connected to a grounded lamp 413 whichaccordingly will light when switch 397 is closed to indicate the closedcondition of such last switch. The other pole 411 energizes groundedcoil 415 of relay assembly 417. The latter assembly includes theaforementioned relays 347 and 385 and an additional and normally closedrelay 419. Accordingly, when coil 415 is energized by reason of closureof switch 397, switch 386 of relay 385 will be magnetically moved toclosed condition while the switches 349 and 420 of relays 347 and 419will be magnetically moved to open condition. Relay 419 is connected inseries with the circuit of the centrifuge motor (not shown) andaccordingly when it opens the centrifuge will stop. Relay 347, becauseof its interposition between terminal 287 and coil 345, will interruptthe circuit to said last coil when open. Accordingly, magnetically heldswitch 339 will be released and current flow to lamp 360 will terminate.In consequence lamp 360 will extinguish, relays 369 and 371 will openand relays 297 and 303 will be restored to a closed position. As aresult, pump action will stop (because relay 371 is open); solenoid 239will be deenergized (because relay 369 is open) and solenoids 241 and243 will become deenergized (by reason of the opening of the respectiveof relays 297 and 303).

By reason of the foregoing construction controlled washing volumes canbe forced into each of containers 37 and 38 notwithstanding differentresistances to flow in said containers. In prior devices while the totalwash volume can be controlled, the volume to each container isuncontrollable because differential resistance in the containers causesthe paths of wash fluid which comes from a common pressure head to movein an uncontrolled fashion. In consequence of the invention,simultaneous washing of a plurality of batches of particles can beachieved in a period substantially no longer than the time required forwashing a single batch. Thereby, for the first time there is provided awashing centrifuge assembly practical for simultaneous processing of aplurality of batches of materials.

The process described has been practiced effectively in reconstitutingthawed red blood cells which theretofore had been preserved by freezingand protected from freeze damage by various cryoprotective agents. Amongthe latter which have been removed are glycerin, an intracellular agentused in high and low concentrations, and polyvinyl pyrrolidone, anextracellular agent.

For removal of glycerinating agent from highly glycerinated frozen redblood cells, a pair of batches, each of which comprises the yield of 500milliliters of whole blood, is first thawed and thereafter spun in ahigh speed centrifuge for a period sufficient for separating the cellsand available liquid. The supernatant is then removed and the red bloodcells of each batch are transferred to one of containers 37 and 38,respectively, for processing in accordance with the invention.

A first wash solution, preferably an aqueous manitol-saline, forosmotically shrinking the red blood cplls and thereby increasing theirdensity, is first forced through each batch by independent pressuremeans for a period sufficient to condition the cells for readyseparation from environmental fluid when centrifuged. Thereafter, asecond wash solution having a decrea neg concentration or gradient ofcell shrinking agent is used to gradually decrease the level of cellularshrinkage to a nullity as a density differential between cells andenvironmental fluid can be maintained under centrifugal force withoutsaid agent. After the manitol has been completely dissipated the cellswill restore to normal size and washing may be completed with hypertonicwash solution of about 5 percent glucose in normal saline. The foregoingprocedure suitably reconstitutes frozen red blood cells for use intransfusion.

The hypertonic solution may omit the 5 percent glucose in which eventthe decreasing manitol gradient may be established with isotonic saline.Moreover, if desired, the original wash solution may be omitted and allwashing may be done with normal saline or the hypertonic solution. Insuch event, however, great care should be exercised to avoid high redcell loss during initial periods of washing. Such likelihood isminimized by manitol in the first wash solution which causes an osmoticsqueeze on the red cells. Thereby, cell density is increased enablingimproved separation of red cells from the less dense glycerinating agentfor removal with effluent.

As many substitutions or changes could be made in the above describedconstruction and process, and as many apparently widely differentembodiments of the invention within the scope of the claims could beconstructed without departing from the scope and spirit thereof, it isintended that all matter contained in the accompanying specificationshall be interpreted as being illustrative and not in a limiting sense.

We claim:

1. In a washing centrifuge assembly having a centrifuge rotor and a pairof containers supported from said centrifuge rotor for rotationtherewith, a combination comprising: charging means for moving influentin predetermined volumes into each of said containers under anindependent pressure head as said rotor spins for washing thereincontained material. in cluding means defining an independent influentpath for the influent for each of said containers; and discharging meansfor collecting effluent from each of said containers in a volumecorresponding to influent moved into said containers.

2. A combination according to claim 1 in which the charging meanscomprises a pump for substantially simultaneously effecting flow ofindependent substantially equal influent volumes into said containers,respectively, and means defining an influent pathway between said pumpand each of said containers.

3. A combination according to claim 1 in which the means defining anindependent influent path includes distributor means including a partarranged for corotation with said rotor and having influent outletmeans, said distributor means having influent inlet means, and means forisolating between said inlet means and said 'outlet means all influentfor one con tainer from all influent for the other.

4. A combination according to claim 1 characterized by distributor meansarranged in fluid association with said charging means for deliveringinfluent to said containers and having first and second parts arrangedfor relative rotation, one of said parts arranged for corotation withsaid rotor; influent inlet and outlet means associated with said firstand second parts, and chamber defining means arranged in saiddistributor means for isolating between said inlet and outlet openingsall influent for each chamber from all influent for the other chamber.

5. A combination according to claim 1 further characterized by adistributor-collector mounted for fluid communication with said chargingmeans and said discharging means, said distributor-collector havingfirst and second parts arranged for relative rotation; influent outletand effluent inlet means associated with said second part; and meansarranged between said first and second parts defining a plurality ofchambers for isolating all influent for each container from all theinfluent for the other container and for isolating all effluent from allinfluent.

6. A combination according to claim 5 in which the chambers forisolating the influent are of lowermost disposition in saiddistributor-collector for minimizing contamination of influent witheffluent.

7. A combination according to claim 1 further characterized by aninfluent reservoir disposed above said containers for deliveringinfluent to said charging means; an influent conduit arranged in fluidcommunication with said charging means and defining a first influentpath to each of said containers, and a tube arranged in fluidcommunication with said reservoir and defining a second influent pathbypassing said charging means for gravity feed to each of saidcontainers through an associated of said influent conduits.

8. A combination according to claim 7 characterized by an electricallyenergized flow control mechanism associated with said paths forestablishing fluid communication between said containers and selectedsaid charging means and reservoir.

A combination according to claim 8 in which the flow control mechanismincludes means for simultaneously clamping said conduits upstream oftheir connection to said tubes and opening said tubes, andsimultaneously clamping said tubes and opening said conduits.

iii. A combination according to claim 8 further characterized byeffluent duct means arranged in fluid communication with saiddischarging means for removal of effluent form said containers, saidflow control mechanism arranged for stopping and permitting flow in saideffluent duct means simultaneously with the interruption and permittingof flow in said influent conduits upstream of the connection thereof tosaid tubes.

1!. A combination according to claim 10 in which said flow controlmechanism comprises a plurality of pressure members spaced from saidcontainers for simultaneously closing said conduits and duct meanssupported for gravity filling said containers, conduits and duct meansbetween said pressure members.

12. In a washing centrifuge assembly having a rotor, container meanssupported from said rotor for rotation therewith, charging means formoving influent into said container means as said rotor spins,discharging means for receiving effluent from said container meansduring movement of and in substantially the same volume as influentmoved, a combination comprising passaged means arranged in fluidassociation with said charging means and discharging means forintroducing influent into and removing effluent from said containermeans, and means responsive to centrifugal force and having a firstcondition in which said passaged means is shut for blocking flow intoand out of said container means and a second condition in which saidpassaged means is open for flow into and out of said container means.

13. A combination according to claim H2 in which the means responsive tocentrifugal force comprises a clamping member normally biased foreffecting said first condition and for moving to effect said secondcondition upon generation in said assembly of centrifugal force ofpredetermined magnitude.

14. A combination according to claim 13 in which said clamping member iscorotational with said rotor and characterized by spring means forbiasing said clamping member for effecting said first condition andproportioned to yield upon generation in said assembly of a centrifugalforce of said predetermined magnitude.

15. in a washing centrifuge assembly having a first rotor, containermeans corotationally supported from said first rotor, charging means formoving influent into said container means as said first rotor spins,discharging means for receiving effluent from said container meansduring movement otand in substantially the same volume as influentmoved, a combina tion comprising: a body corotationally secured to saidfirst rotor for securing said charging and discharging means to saidfirst rotor, and a disposable distributorcollector removably mounted insaid body and arranged medially of said charging and discharging meansfor transferring influent to and effluent from said container meanswhile said first rotor is spinning 16. A combination according to claim15 in which said distributor-collector has a stator and a second rotormounted for rotation about said stator and arranged in keyed associationwith said body, some of said charging and discharging means extendinginto and out of said second rotor for delivery of influent and removalof effluent from said containers, other of said charging and dischargingmeans extending into and out of said stator for delivery of influent toand removal of effluent from said distributor-collector.

17. A combination according to claim 16 in which said body has anopening, said second rotor disposed outwardly from said stator andhaving a depending keying extension removably engaged in said openingfor rotating said second rotor with said first rotor.

18. A combination according to claim 17 in which saiddistributor-collector is vertically disposed with said stator having anextension projecting upwardly from said second rotor and characterizedby a bearing mounted in nonrotating, stationary relation relative saidfirst and second rotors and having means for engaging said extension forholding said stator fixed relative to said first and second rotors whilesaid first and second rotors spin.

19. A combination according to claim 15 in which said body comprises acentrifugal clamp for enabling flow through said passaged means onlyupon generation of centrifugal force of a predetermined level,

20. in a process for material separation by centrifugation during whicha first material is removed from a second material by a wash fluid whichis first passed through the second material and therefrom removed withfirst material as spent fluid, the improvement comprising passingpredetermined volumes of the wash fluid each under an independentpressure head through separate flow paths into each of a plurality ofbatches of said second material while spun about a common axis.

21. A process according to claim 20 in which the wash fluid is forced insubstantially equal volumes through said batches, respectively.

22. A process according to claim 20 in which the wash fluid is forcedthrough said batches substantially simultaneously,

1. In a washing centrifuge assembly having a centrifuge rotor and a pairof containers supported from said centrifuge rotor for rotationtherewith, a combination comprising: charging means for moving influentin predetermined volumes into each of said containers under anindependent pressure head as said rotor spins for washing thereincontained material, including means defining an independent influentpath for the influent for each of said containers; and discharging meansfor collecting effluent from each of said containers in a volumecorresponding to influent moved into said containers.
 2. A combinationaccording to claim 1 in which the charging means comprises a pump forsubstantially simultaneously effecting flow of independent substantiallyequal influent volumes into said containers, respectively, and meansdefining an influent pathway between said pump and each of saidcontainers.
 3. A combination according to claim 1 in which the meansdefining an independent influent path includes distributor meansincluding a part arranged for corotation with said rotor and havinginfluent outlet means, said distributor means having influent inletmeans, and means for isolating between said inlet means and said outletmeans all influent for one container from all influent for the other. 4.A combination according to claim 1 characterized by distributor meansarranged in fluid association with said charging means for deliveringinfluent to said containers and having first and second parts arrangedfor relative rotation, one of said parts arranged for corotation withsaid rotor; influent inlet and outlet means associated with said firstand second parts, and chamber defining means arranged in saiddistributor means for isolating between said inlet and outlet openingsall influent for each chamber from all influent for the other chamber.5. A combination according to claim 1 further characterized by adistributor-collector mounted for fluid communication with said chargingmeans and said discharging means, said distributor-collector havingfirst and second parts arranged for relative rotation; influent outletand effluent inlet means associated with said second part; and meansarranged between said first and second parts defining a plurality ofchambers for isolating all influent for each container from all theinfluent for the other container and for isolating all effluent from allinfluent.
 6. A combination according to claim 5 in which the chambersfor isolating the influent are of lowermost disposition in saiddistributor-collector for minimizing contamination of influent witheffluent.
 7. A combination according to claim 1 further characterized byan influent reservoir disposed above said containers for deliveringinfluent to said charging means; an influent conduit arranged in fluidcommunication with said charging means and defining a first influentpath to each of said containers, and a tube arranged in fluidcommunication with said reservoir and defining a second influent pathbypassing said charging means for gravity feed to each of saidcontainers through an associated of said influent conduits.
 8. Acombination according to claim 7 characterized by an electricallyenergized flow control mechanism associated with said paths forestablishing fluid communication between said containers and selectedsaid charging means and reservoir.
 9. A combination according to claim 8in which the flow control mechanism includes means for siMultaneouslyclamping said conduits upstream of their connection to said tubes andopening said tubes, and simultaneously clamping said tubes and openingsaid conduits.
 11. A combination according to claim 10 in which saidflow control mechanism comprises a plurality of pressure members spacedfrom said containers for simultaneously closing said conduits and ductmeans supported for gravity filling said containers, conduits and ductmeans between said pressure members.
 11. A combination according toclaim 8 further characterized by effluent duct means arranged in fluidcommunication with said discharging means for removal of effluent formsaid containers, said flow control mechanism arranged for stopping andpermitting flow in said effluent duct means simultaneously with theinterruption and permitting of flow in said influent conduits upstreamof the connection thereof to said tubes.
 12. In a washing centrifugeassembly having a rotor, container means supported from said rotor forrotation therewith, charging means for moving influent into saidcontainer means as said rotor spins, discharging means for receivingeffluent from said container means during movement of and insubstantially the same volume as influent moved, a combinationcomprising passaged means arranged in fluid association with saidcharging means and discharging means for introducing influent into andremoving effluent from said container means, and means responsive tocentrifugal force and having a first condition in which said passagedmeans is shut for blocking flow into and out of said container means anda second condition in which said passaged means is open for flow intoand out of said container means.
 13. A combination according to claim 12in which the means responsive to centrifugal force comprises a clampingmember normally biased for effecting said first condition and for movingto effect said second condition upon generation in said assembly ofcentrifugal force of predetermined magnitude.
 14. A combinationaccording to claim 13 in which said clamping member is corotational withsaid rotor and characterized by spring means for biasing said clampingmember for effecting said first condition and proportioned to yield upongeneration in said assembly of a centrifugal force of said predeterminedmagnitude.
 15. In a washing centrifuge assembly having a first rotor,container means corotationally supported from said first rotor, chargingmeans for moving influent into said container means as said first rotorspins, discharging means for receiving effluent from said containermeans during movement of and in substantially the same volume asinfluent moved, a combination comprising: a body corotationally securedto said first rotor for securing said charging and discharging means tosaid first rotor, and a disposable distributor-collector removablymounted in said body and arranged medially of said charging anddischarging means for transferring influent to and effluent from saidcontainer means while said first rotor is spinning.
 16. A combinationaccording to claim 15 in which said distributor-collector has a statorand a second rotor mounted for rotation about said stator and arrangedin keyed association with said body, some of said charging anddischarging means extending into and out of said second rotor fordelivery of influent and removal of effluent from said containers, otherof said charging and discharging means extending into and out of saidstator for delivery of influent to and removal of effluent from saiddistributor-collector.
 17. A combination according to claim 16 in whichsaid body has an opening, said second rotor disposed outwardly from saidstator and having a depending keying extension removably engaged in saidopening for rotating said second rotor with said first rotor.
 18. Acombination according to claim 17 in which said distributor-collector isvertically disposed with said stator having an extension projectingupwardly from said second rotor and characterized by a bearing mountedin nonrotating, stationary relation relative said first and secondrotors and having means for engaging said extension for holding saidstator fixed relative to said first and second rotors while said firstand second rotors spin.
 19. A combination according to claim 15 in whichsaid body comprises a centrifugal clamp for enabling flow through saidpassaged means only upon generation of centrifugal force of apredetermined level.
 20. In a process for material separation bycentrifugation during which a first material is removed from a secondmaterial by a wash fluid which is first passed through the secondmaterial and therefrom removed with first material as spent fluid, theimprovement comprising passing predetermined volumes of the wash fluideach under an independent pressure head through separate flow paths intoeach of a plurality of batches of said second material while spun abouta common axis.
 21. A process according to claim 20 in which the washfluid is forced in substantially equal volumes through said batches,respectively.
 22. A process according to claim 20 in which the washfluid is forced through said batches substantially simultaneously.